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Template:Team:SydneyUni Australia/Calendar/Events List
From 2013.igem.org
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title: 'Colony Screening', | title: 'Colony Screening', | ||
start: new Date(2013, 7, 16), | start: new Date(2013, 7, 16), | ||
| - | end: new Date(2013, | + | end: new Date(2013, 7, 18), |
description: '<b>Members:</b> Rob, Nick, Desmond, Andrew<br> <b>What we did: </b> - Checked plates and learned that transforming our Gibson Assembly product into TOP10 (rather than EPI300) worked better than before.<br><br>- This was confirmed on Saturday morning (17th) after re-transforming the same reaction with the last of our Gibson Assembly product, in order to obtain more colonies for screening (for finding which of the colonies contains the correctly assembled pathway).<br><br>- A master plate containing all colonies was patched for phenotypic screening the following week.' | description: '<b>Members:</b> Rob, Nick, Desmond, Andrew<br> <b>What we did: </b> - Checked plates and learned that transforming our Gibson Assembly product into TOP10 (rather than EPI300) worked better than before.<br><br>- This was confirmed on Saturday morning (17th) after re-transforming the same reaction with the last of our Gibson Assembly product, in order to obtain more colonies for screening (for finding which of the colonies contains the correctly assembled pathway).<br><br>- A master plate containing all colonies was patched for phenotypic screening the following week.' | ||
}, | }, | ||
| Line 150: | Line 150: | ||
description: '<b>Members:</b> Viv<br> <b>What we did: </b>Treated plasmids with RNAse, repeated final steps of plasmid prep. ' | description: '<b>Members:</b> Viv<br> <b>What we did: </b>Treated plasmids with RNAse, repeated final steps of plasmid prep. ' | ||
}, | }, | ||
| + | // -----------------Week 10 ---------------- | ||
| + | { | ||
| + | title: 'Chloride Assay Prep', | ||
| + | start: new Date(2013, 7, 24), | ||
| + | end: new Date(2013, 7, 25), | ||
| + | description: '<b>Members:</b> Andrew, Nick <br> <b>What we did: </b>- Plated out cells carrying pUC19-dhlB for positive control in [Cl-] assay. <br><br>- Inoculated 4ml broths for [Cl-] assay' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Chloride Assay', | ||
| + | start: new Date(2013, 7, 26), | ||
| + | description: '<b>Members:</b> Desmond<br> <b>What we did: </b>- Transferred overnight cultures (4mL) to fresh 50 mL LB and grown to an OD of ~1.0. Cells were harvested, cleaned and resuspended in 2mM chloroacetate for overnight incubation for chloride assay the next day.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Chloride Assay Stuff', | ||
| + | start: new Date(2013, 7, 27), | ||
| + | description: '<b>Members:</b> Rob<br> <b>What we did: </b>Remaining E.coli was spread plated onto Mackonkey agar for future negative Cl- assays.<br><br>Solutions for plasmid preps and chloride assays were made. We suspect the ambiguous plasmid prep and chloride assays results are due to poor stocks.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Chloride Assay', | ||
| + | start: new Date(2013, 7, 28), | ||
| + | description: '<b>Members:</b> Rob<br> <b>What we did: </b>Inoculated LB-Cm with our clones [2,10,38, 42,54,64, rfp(63)], LB-Am (positive control) and LB (negative control) for plasmid prep and chloride assay. No growth was observed in positive and negative control so no culture could be transferred for chloride assay.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Chloride Assay Stuff', | ||
| + | start: new Date(2013, 7, 29), | ||
| + | description: '<b>Members:</b> Rob, Shuravi, Desmond<br> <b>What we did: </b>Clones grown in LB-antibiotic broth overnight were washed and added to 2mM DCA reagents and another set in 2mM chloroacetate reagent. Standards using NaCl solution was made simultaneously to ensure accuracy of the assay.<br><br>Plasmid preps were done using clones 2, 10, 38, 54, 42, 64 and rfp 63.<br><br>The samples were patch plated onto new LB-Cm plates.' | ||
| + | }, | ||
| + | // -----------------Week 11 ---------------- | ||
| + | { | ||
| + | title: 'Diagnostic Gel', | ||
| + | start: new Date(2013, 7, 31), | ||
| + | description: '<b>Members:</b> Rob <br> <b>What we did: </b>A diagnostic digest was set up for clones 42, 54 and 64 using EcoRV and 2, 38, rfp using EcrIVHF. Banding patterns on our gel was difficult to see but was better visualised under UV.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'PCR Work', | ||
| + | start: new Date(2013, 8, 2), | ||
| + | description: '<b>Members:</b> Rob, James <br> <b>What we did: </b>Our new primers arrived. Today we PCR screened for dhlB and found the reverse primer corresponded to the synthetic dhlB sequence. Our PCR products were then loaded onto gel. The results were a little disappointing as there was no banding on our first gel and the ladder was difficult to see. Primer dimers were observed on our gel.<br><br>We also inoculated another 20 clones for screening from our initial Gibson Assembly transformation of p-p [contains p450] and p-a [contains adh] (These are our two different pathways).<br><br>We prepared some competent EPI400 cells. These are to be used later for the transformation of our GA products.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Cyril's Birthday', | ||
| + | start: new Date(2013, 8, 2), | ||
| + | description: '<b>Members:</b> Cyril <br> <b>Happy Birthday :) </b>' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Cell Transformation, Gibson Assembly', | ||
| + | start: new Date(2013, 8, 3), | ||
| + | description: '<b>Members:</b> Andrew, Rob <br> <b>What we did: </b>We transformed our Epi400 cells with our Gibson assembly products. Results did not look very promising. The Epi400 cells looked as they did when we transformed Epi300. The control worked and few cells grew with the positive Gibson assembly. There was more growth of cells transformed with p-p and p-a.<br><br>Another Gibson Assembly was set up for transformation. We found iGEMblock1 did not contain enough DNA leaving less than needed for the p-a sample.<br><br>We PCR screened for dhlB in colonies 100-139 however did not have a positive control (did not work yesterday). A gel was run for the dhlB PCR products.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Cell Transformation', | ||
| + | start: new Date(2013, 8, 4), | ||
| + | description: '<b>Members:</b> Rob <br> <b>What we did: </b>Another transformation was performed using the second set of Gibson assembly products into E.coli TOP 10 and E.coli Epi400. The transformed cells were plated onto the appropriate antibiotic media. ' | ||
| + | }, | ||
| + | { | ||
| + | title: 'PCR and Diagnostic Gel', | ||
| + | start: new Date(2013, 8, 5), | ||
| + | description: '<b>Members:</b> Andrew <br> <b>What we did: </b>We did a PCR of dhlB from the GA reaction products using three samples (p-p, p-a and a negative control containing no DNA)<br><br>All GA enzymes were heat-killed by placing using thermocycler at 95degres.<br><br>We ran a gel of dhlB PCR of Gibson assembly products. The resulting bands were clear and as expected.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Diagnostic Gel', | ||
| + | start: new Date(2013, 8, 6), | ||
| + | description: '<b>Members:</b> Rob <br> <b>What we did: </b>A diagnostic digest was performed on our old plasmid preps. p-p was digested with BamH1, MluI, SmaI, SphI and Sal1. P-a digested with MluI, SmaI, SphI and rfp had no restriction enzyme added' | ||
| + | }, | ||
| + | // -----------------Week 12 ---------------- | ||
| + | { | ||
| + | title: 'Digests', | ||
| + | start: new Date(2013, 8, 9), | ||
| + | description: '<b>Members:</b> Andrew, Desmond, Rob <br> <b>What we did: </b>Two digests were set up in parallel (p-p and p-a) using SspI and later purified using Quiaquick columns.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Plasmid Prep, PCR Screen', | ||
| + | start: new Date(2013, 8, 10), | ||
| + | description: '<b>Members:</b> Andrew <br> <b>What we did: </b>A plasmid prep was performed on pSB-RFP, taken from transformed E.coli TOP10 cells. Stopped at precipitation stage.<br><br>We received our new set of primers! We used these to PCR screen all our selected Gibson assembly plasmids and GA restriction products and our positive GA control (puc19-dhla-dhlb)<br><br>To confirm that our PCR screening had worked we ran a gel of all our PCR products' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Plasmid Prep, Gradient PCR', | ||
| + | start: new Date(2013, 8, 11), | ||
| + | description: '<b>Members:</b> Vivian, James, Desmond, Andrew <br> <b>What we did: </b>A gradient PCR was done on dhlB and dhlA from our positive GA samples.<br><br>Gels were run for each of our PCR products from the previous day.<br><br>The plasmid prep from earlier in the week was completed. Andrew later digested the Gibson assembly product using sspI. Desmond set up a digest for Rfp using ECRo1 and ran a gel of the digests.<br><br>Another PCR was done using the Gibson assembly digested fragments.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Ligation and Transformation', | ||
| + | start: new Date(2013, 8, 12), | ||
| + | description: '<b>Members:</b> Rob, Andrew, Vivian <br> <b>What we did: </b>Today a digestion of pSB-rfp was made using the enzymes, xbaI and PstI for ligation. Our PCR fragments (dhlA and dhlB) were also digested using the same restriction enzymes.<br><br>Following purification of our PCR DNA we ligated our inserts (dhlA, dhlB or dhla-dhlb) into our plasmid vector (pSB). We split the reaction volume to two and use one later by storing in the cool room.<br><br>The ligated products were transformed into E.coli Top10 and EpI300 and plated on to LB-Cm.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'PCR Screen', | ||
| + | start: new Date(2013, 8, 13), | ||
| + | description: '<b>Members:</b> Rob <br> <b>What we did: </b>We PCR screened for the correct dhlA, dhlB, dhlA-dhlB inserts by creating patch plates of white colonies on LB-Cm. Each clone was then mixed with EB and boiled to release DNA from cells. The cell suspension of each clone were used to PCR the DNA. Unfortunately some resulted in no PCR product.' | ||
| + | }, | ||
| + | { | ||
| + | title: 'Plasmid Prep, More Screening', | ||
| + | start: new Date(2013, 8, 14), | ||
| + | description: '<b>Members:</b> Rob, Andrew <br> <b>What we did: </b>Based on yesterday screens we selected clones for plasmid prepping. Plasmids were digested using EcrOV to check length and concentration of DNA.<br><br>To find successfully ligated dhlB we did more PCR screening. This time however we used EPI300 cells instead of TOP10. Screening was also done for dlb-dhla.' | ||
| + | }, | ||
| + | // -----------------Week 13 ---------------- | ||
| + | { | ||
| + | title: 'Gel and Plasmid Prep (Again)', | ||
| + | start: new Date(2013, 8, 9), | ||
| + | description: '<b>Members:</b> Rob, Andrew, James <br> <b>What we did: </b>Following on from yesterdays digested plasmid prep a gel was run to at different concentrations.<br><br>We found, from the gel of our digest, that the plasmids were not concentrated enough.<br><br>Another plasmid prep, using some different clones was done. This time however, we incubated cells overnight to increase plasmid yield.<br><br>We digested and ran a gel of our plasmid for confirmation. The gel turned out perfect!' | ||
| + | }, | ||
| + | |||
{ | { | ||
title: 'Click for Google', | title: 'Click for Google', | ||
Revision as of 13:41, 25 September 2013
